Restriction Fragment Length Polymorphisms (RFLPs) By Amr S. Moustafa, M.D.; Ph.D. Assistant Prof. & Consultant, Medical Biochemistry Dept. College of Medicine, KSU

Objectives  Polymorphisms Vs mutations  RFLPs  Single nucleotide polymorphisms (SNPs)  Minisatellites and microsatellites  Variable number tandem repeat (VNTR)  Medical applications

Objectives  Polymorphisms Vs mutations  RFLPs  Single nucleotide polymorphisms (SNPs)  Minisatellites and microsatellites  Variable number tandem repeat (VNTR)  Medical applications

Background information: Facts  Human DNA sequence is nearly 99.9% identical  Each new zygote contains ~ 100 bp changes not present in the genome of either parents  Only one bp varies between 2 persons per 1500 bp DNA segment  Human genetic diversity manifested as Changes in DNA sequences Protein variations Diseases

Background information: Definitions Alleles: Different forms of the same gene on a specific locus Genotype: The set of alleles that make up the genetic constitution Phenotype: The observable expression of a genotype

Polymorphisms Vs Mutations Genetic polymorphisms: Common alleles > 1% Mutations: Rare alleles < 1%

Polymorphisms: Sites Inter-genes or intronic: Detected by DNA sequence analysis Gene coding sequences: Different protein variants Distinct phenotypes (may be) DNA regulatory regions: may affect phenotypes

Polymorphisms: Detection – 1 At protein level ABO System: A, B & O alleles: Chromosome 9 A & B: 4 nucleotide differences O: One base deletion and frame-shift Phenotypes: O, A, B & AB Rh System: Rh-D: Chromosome 1 Phenotypes: Rh-positive and Rh-negative Incompatibility:Hemolytic disease of newborn

Polymorphisms: Detection – 2 At DNA level  DNA sequence analysis Inherited variations in DNA sequences Different sizes of DNA fragments Restriction enzyme  Restriction fragment length polymorphisms (RFLPs):

Before the Test To whom who knows the answer before Please Let me Look at Think about Answer by myself

The Test Madam I am Adam كل في فلك

ِ After the Test 5’- C T G C A G - 3’ 3’- G A C G T C - 5’ Pst I

RFLPs: Causes Single nucleotide polymorphisms (SNPs): Gain or loss of a restriction site more frequent than mini- & micro-satellites Variable number tandem repeat (VNTR): Alteration of number of nucleotides between restriction sites: Minisatellites: bp Microsatellites: bp 2 unrelated individuals: different pattern Identical twins: identical pattern

RFLPs: VNTR For each person a pair of homologous chromosomes is shown

RFLPs: Medical applications  Mapping a gene to a particular region of a chromosome  Tissue typing for organ transplantation  Paternity testing and forensic applications  Prenatal diagnosis of genetic diseases

RFLPs: Prenatal Diagnosis of sickle cell anemia - 1

RFLPs: Prenatal Diagnosis of sickle cell anemia - 2

RFLPs: SA Genotypes bp Detection of SA gene by PCR

RFLPs: SA Genotypes bp A1A1A2A2 Detection of PstI RFLP of SA gene by PCR